Synthesis, Physicochemical and Biological Study of Gallium-68- and Lutetium-177-Labeled VEGF-A165/NRP-1 Complex Inhibitors Based on Peptide A7R and Branched Peptidomimetic

Neuropilin-1 (NRP-1) is a surface receptor found on many types of cancer cells. The overexpression of NRP-1 and its interaction with vascular endothelial growth factor-165 (VEGF165) are associated with tumor growth and metastasis. Therefore, compounds that block the VEGF165/NRP-1 interaction represent a promising strategy to image and treat NRP-1-related pathologies. The aim of the presented work was to design and synthesize radioconjugates of two known peptide-type inhibitors of the VEGF165/NRP-1 complex: A7R peptide and its shorter analog, the branched peptidomimetic Lys(hArg)-Dab-Pro-Arg. Both peptide-type inhibitors were coupled to a radionuclide chelator (DOTA) via a linker (Ahx) and so radiolabeled with Ga-68 and Lu-177 radionuclides, for diagnostic and therapeutic uses, respectively. The synthesized radioconjugates were tested for their possible use as theranostic-like radiopharmaceuticals for the imaging and therapy of cancers that overexpress NRP-1. The obtained results indicate good efficiency of the radiolabeling reaction and satisfactory stability, at least 3t1/2 for the 68Ga- and 1t1/2 for the 177Lu-radiocompounds, in solutions mimicking human body fluids. However, enzymatic degradation of both the studied inhibitors caused insufficient stability of the radiocompounds in human serum, indicating that further modifications are needed to sufficiently stabilize the peptidomimetics with inhibitory properties against VEGF165/NRP-1 complex formation.


Introduction
NRP-1 plays one of the most important roles in the development of angiogenesis, which is the formation of new blood vessels from existing ones. One of the main stages of angiogenesis is the interaction between the pro-angiogenic factor (VEGF-A 165 ), its receptor (VEGFR-2), and the NRP-1 co-receptor [1][2][3][4][5][6][7]. NRP-1 acts in two ways: it binds to the pro-angiogenic ligand VEGF-A 165 through its b1/b2 subdomains, and in parallel, it acts as a co-receptor for VEGFR-2. The resulting ternary VEGF-A 165 /VEGFR-2/NRP-1 complex induces autophosphorylation of the VEGFR-2 tyrosine kinase domains, which influences cell proliferation, differentiation, migration, gene expression, and apoptotic survival of endothelial cells, which ultimately induces angiogenesis [8][9][10]. Many reports also indicate that NRP-1, which is found on many types of cancer cells, lacks catalytic activity and may also serve as a separate receptor for VEGF-A 165 , stimulating tumor growth and metastasis. NRP-1 overexpression may also increase tumor growth and is often associated with poor prognosis, especially in tumors of epithelial origin [3,[11][12][13][14][15][16][17][18]. Due to the significant role of NRP-1 in angiogenesis and the relatively well-known mechanisms of the formation and Unless otherwise specified, reagents and solvents were obtained from commercial sources and used without further purification. Fmoc-Arg(Pbf)-Wang resin was obtained from Activotec (Cambridge, UK). Amino acids and coupling reagents were purchased from Iris Biotech (Marktredwitz, Germany). DOTA-tris(tBu)-NHS was purchased from CheMatech (Dijon, France). Pooled human serum (HS) was obtained from Innovative Research (Novi, MI, USA).
Deionized water was prepared in a Hydrolab water purification system (Hydrolab, Straszyn, Poland).
The cleavage of the final conjugates were performed using the mixture of TFA:PhOH:H2O:TIPS (88:5:5:2, v/v/v/v) for 2 h. Crude conjugates were precipitated by a dropwise addition into a cold diethyl ether, and then were purified using semipreparative KNAUER RP-HPLC in System 1a for conjugate 1 and System 2a for conjugate 2. Purified conjugates were analyzed by mass spectrometry method (ESI-MS). In some cases, we also added 0.5-2.0 µL 0.1 M HCl to obtain a desired pH. The reaction mixture at pH 4.5 was heated for 10 min at 95 • C. The resulting conjugates were then purified by RP-HPLC in System 3 with gamma detection for radioconjugate 177 Lu-1 (RCY 95.5 ± 1.2%, n = 4, molar activity 0.3 MBq/nmol, pH 4.5) and in System 4 with gamma detection for radioconjugate 177 Lu-2 (RCY 96.2 ± 2.6%, n = 4, molar activity 0.3 MBq/nmol, pH 4.5). Pure fractions of 177 Lu-1 and 177 Lu-2 radioconjugates were evaporated under N 2 and dissolved on PBS.
All radioconjugates were purified before the use in further experiments, namely, the lipophilicity and stability studies.
Ga-DOTA-Ahx-A7R (Ga-1) and Lys(hArg)-Dab(Ahx-DOTA-Ga)-Pro-Arg (Ga-2) cold reference compounds were synthesized according to the following procedure: into a vial containing approximately 70 nmol of conjugate 1 or 2 dissolved in 300 µL of 0.2 M acetate buffer (pH 4.5), we added the 60 µL of 1.34 mg/mL GaCl 3 solution in 0.065 M HCl. The reaction mixture at pH 4.0 was heated for 10 min at 95 • C. The reaction progress was checked by RP-HPLC in System 3 for Ga-1 and in System 4 for Ga-2.
Lu-DOTA-Ahx-A7R (Lu-1) and Lys(hArg)-Dab(Ahx-DOTA-Lu)-Pro-Arg (Lu-2) cold reference compounds were synthesized according to the following procedure: to a vial containing approximately 70 nmol of conjugate 1 or 2 dissolved in 600 µL of 0.2 M acetate buffer (pH 4.5), we added the 3.5 µL of concentrated LuCl 3 solution in 14 M HCl. The reaction mixture at pH 4.0 was heated for 10 min at 95 • C. The reaction progress was checked by RP-HPLC in System 3 for Lu-1 and in System 4 for Lu-2.

Physicochemical Properties Study of the Radioconjugates
All studies of the physicochemical properties of the synthesized radiopreparations were carried out using radioconjugates previously isolated from the reaction mixture.
Radioconjugates lipophilicity test The lipophilicity (L) of tested radiocompounds, defined as the decimal logarithm (logP) of the partition coefficient (P) of the compound between the two immiscible phases, was determined in the system PBS solution (aqueous phase, polar phase, pH 7.4) and n-octanol (organic, non-polar phase) according to the following formula: where A o -organic phase radioactivity; A w -aqueous phase radioactivity. Radioactivity of both phases, resulting from the concentration of the tested radiocompound in each of them, was determined by measuring the gamma radiation with a Wizard counter in three independent experiments.
Before starting the research, both liquid phases were saturated with each other to avoid errors resulting from the mutual solubility of both liquids. The partition coefficient (P) result is shown as the mean ± SD. After the end of the experiment, we performed RP-HPLC analysis of the aqueous phase to confirm the tested radioconjugate remained intact during the experiment.
Radioconjugates stability tests In accordance with requirements for potentially novel radiopharmaceuticals, we tested newly designed radiopreparations for their stability in solutions that act as human body fluids and also in human serum [54,55].
Stability studies in PBS buffer, cysteine and histidine Stability studies in PBS buffer were performed in order to investigate the possible influence of buffer components on the radiocompound decomposition process. We isolated 68 Ga-1, 177 Lu-1, 68 Ga-2, and 177 Lu-2 radioconjugates from the reaction mixture and incubated each in PBS buffer. After about 3 h, we analyzed aliquots by RP-HPLC in System 3 with gamma detection for 68 Ga-1 and 177 Lu-1 and in System 4 with gamma detection for 68 Ga-2 and 177 Lu-2.
Stability studies of the isolated from the reaction mixture 68 Ga-1, 177 Lu-1, 68 Ga-2, and 177 Lu-2 radioconjugates in solutions containing excess amounts of strongly competing natural ligands with chemically reactive groups, e.g., -NH 2 , -SH, and -COOH (the so-called challenge experiments). For this purpose, each radioconjugate was incubated at 37 • C in 1 mM cysteine (Cys) or histidine (His) solutions in PBS buffer (the ligand concentration was about 1000 times higher than the concentration of tested radioconjugate). After the specified incubation time, which was up to 4 h for the 68 Ga-radiocompounds and up to 6 days for the 177  Stability studies in human serum Stability studies of the 68 Ga-1, 177 Lu-1, 68 Ga-2, and 177 Lu-2 radioconjugates in human serum (HS) were performed according to the following procedure: into a vial containing 900 µL of HS we added 100 µL of the tested radioconjugate and incubated the mixture at 37 • C. After the specified incubation times (10 min-4.5 h for 68 Ga-radiocompounds and 10 min-4 days for 177 Lu-radiocompounds), we withdrew the aliquots, mixed with ethanol to precipitate the proteins, and centrifuged (14,000 rpm, 5-15 min) to separate the protein compounds from the supernatant. The gamma radioactivity of the obtained precipitate and supernatant was then measured using a Wizard detector. Using the formula below, the amount of radioconjugate remaining in the supernatant (and also bound by serum protein components) was calculated.
∑ liquid phase radioactivity ∑ liquid phase radioactivity + ∑ percipitate radioactivity × 100% We also analyzed the supernatant at each time by RP-HPLC in System 3 with gamma detection for 68 Ga-1 and 177 Lu-1 and System 4 with gamma detection for 68 Ga-2 and 177 Lu-2 to check whether the studied radiocompound remained unchanged during the experiment.

Syntheses of Conjugates 1 and 2
The synthesis of conjugates 1 and 2 (Scheme 1) was carried out by Solid Phase Peptide Synthesis (SPPS) on the preloaded Fmoc-Arg(Pbf)-Wang resin following the Fmoc chemistry according to the standard coupling DIC/HOBt protocol. In the case of synthesis of conjugate 1, after building a linear peptide on the resin and coupling of the Ahx spacer, the chelator DOTA was attached by the active ester method using DOTA-tris(tBu)-NHS.
The synthesis of conjugate 2 required a few additional steps, as shown in Scheme 1. After the synthesis of fully protected, linear tripeptide (Fmoc-Dab(Alloc)-Pro-Arg(Pbf)-Wang), we attached the N-terminal lysine using Boc-Lys(Fmoc), which enabled the attachment of another Boc-Lys(Fmoc) to the side chain after removal of the Fmoc group from the epsilon amino group. After Fmoc deprotection of the epsilon amino group of the second lysine, we carried out the guanylation reaction for 4 days to lead to hArg creation using di-Boc-S-methylisothiourea, until the Kaiser test was negative. Next, after Alloc deprotection of the amino group of the side chain of the Dab residue, we coupled Fmoc-Ahx. Following the coupling sequence, Fmoc deprotection was performed as the next step, and then the DOTA chelator was attached by the active ester method using DOTA-tris(tBu)-NHS. The cleavage of the synthetized compounds from the resin was performed using the TFA:PhOH:H 2 O:TIPS mixture simultaneously removing all Boc protections. We purified the crude compounds by RP-HPLC and analyzed by ESI-MS (Table 1)

Synthesis of Cold References Compounds
RP-HPLC analyses of the reaction mixtures of the cold reference compounds are presented in Figure 3.  The results of ESI-MS analyses confirming the cold reference compounds were The results of ESI-MS analyses confirming the cold reference compounds were obtained are shown in Table 3.

Lipophilicity Studies
Lipophilicity is one of the most important physicochemical parameters affecting the absorption, distribution, metabolism, and excretion of drug molecules in the organism, the so-called ADME profile [56][57][58][59]. Preparations characterized by high lipophilicity show a high affinity for fats and a low affinity for water; they are also able to effectively penetrate cell membranes. From the point of view of pharmacology, lipophilicity is an important prognostic factor for drugs and other medical preparations to predict toxic activity and to characterize biological activity, the ability to accumulate in organisms, and the metabolism of substances [60][61][62][63][64][65]. The lipophilicity values obtained for tested radioconjugates 68 Ga-1, 177 Lu-1, 68 Ga-2, and 177 Lu-2 (presented as the mean ± SD) are shown in Table 4. The main reason for the low lipophilicity value is that the tested radiocompounds are based on peptides and peptidomimetics, which are usually characterized by a low lipophilicity value. Moreover, conjugation of the macrocyclic DOTA chelator with the biomolecule also results in a lower lipophilicity value of the conjugate compared to the lipophilicity of the biomolecule itself. Radiochromatograms of the aqueous phases after the lipophilicity tests of the radiopreparations showed, in all cases, single peaks at R T values that corresponded to the tested radiocompounds, which proves the stability of the radiopreparations during the experiment.

Stability Studies of Radioconjugates in PBS Buffer, Cysteine and Histidine
Stability tests of all radioconjugates incubated in PBS solution for about 3 h showed no decomposition of radioconjugates. In all the RP-HPLC radiochromatograms only single peaks were recorded, with R T values that corresponded to the appropriate radioconjugate, which confirms the stability of radioconjugates under such conditions.

Stability Tests for 177 Lu-1 and 177 Lu-2 Radioconjugates
Stability studies of the radioconjugates containing lutetium-177 radionuclide in Cys and His solutions were carried out in the period of about 6 days, corresponding to the one half-live of the radionuclide lutetium-177. Incubation of the 177 Lu-1 radioconjugate in Cys and His solutions for a longer period, more than 4 days, showed slow degradation of this radiopreparation in both solutions ( Figure 6). The radioconjugate was completely stable for 1 day, but after 6 days, traces of the products of radioconjugate decomposition were already visible, and the degradation of the radioconjugate was noticeably higher during incubation in the His solution (Figure 6 right).
In contrast, the 177 Lu-2 radioconjugate proved to be completely stable in both solutions throughout the incubation period (Figure 7). and His solutions were carried out in the period of about 6 days, corresponding to the one half-live of the radionuclide lutetium-177. Incubation of the 177 Lu-1 radioconjugate in Cys and His solutions for a longer period, more than 4 days, showed slow degradation of this radiopreparation in both solutions ( Figure 6). The radioconjugate was completely stable for 1 day, but after 6 days, traces of the products of radioconjugate decomposition were already visible, and the degradation of the radioconjugate was noticeably higher during incubation in the His solution (Figure 6 right).
HPLC System 3. HPLC System 3. In contrast, the 177 Lu-2 radioconjugate proved to be completely stable in both solutions throughout the incubation period ( Figure 7).

Stability Studies of Radioconjugates in Human Serum
Radiochromatograms illustrating the results of the stability tests of the 68 Ga-1, 177 Lu-1 and 68 Ga-2, 177 Lu-2 radioconjugates in human serum are shown in Figure 8. The statistical data of the radiochemical purity (presented as the mean ± SD, n = the number of repetitions) of the obtained 68 Ga-1, 177 Lu-1, 68 Ga-2, and 177 Lu-2 radioconjugates in the human serum stability tests are presented in Table 5.   Table 5. Statistical data of the 68 Ga-1, 177 Lu-1, 68 Ga-2, and 177 Lu-2 radioconjugates radiochemical purity corresponding to the radiochromatograms presented in Figure 8.

RCP ± SD, n = 3 (%) after 10 min after 4.5 h after 1 day After 4 days 68 Ga-1
65.3 ± 3.7 36.6 ± 1.4 n/a n/a 68 Ga-2 27.7 ± 2.0 3.6 ± 0.2 n/a n/a 177 Lu-1 39.2 ± 2.0 ------2.6 ± 0.2 177 Lu-2 13.8 ± 1.5 ---2.7 ± 0.1 ---As can be seen, all radioconjugates were unstable in HS due to biodegradation of the A7R peptide and Lys(hArg)-Dab-Pro-Arg peptidomimetic by endogenous enzymes. After 10 min of incubation, the radiochromatograms already showed additional peaks corresponding to the enzymatic degradation products. During incubation, the height of these additional peaks increased, and the peak heights corresponding to the tested radioconjugates decreased. After about 4 h in the case of the 68 Ga-1 and 68 Ga-2  Table 5. Statistical data of the 68 Ga-1, 177 Lu-1, 68 Ga-2, and 177 Lu-2 radioconjugates radiochemical purity corresponding to the radiochromatograms presented in Figure 8. As can be seen, all radioconjugates were unstable in HS due to biodegradation of the A7R peptide and Lys(hArg)-Dab-Pro-Arg peptidomimetic by endogenous enzymes. After 10 min of incubation, the radiochromatograms already showed additional peaks corresponding to the enzymatic degradation products. During incubation, the height of these additional peaks increased, and the peak heights corresponding to the tested radioconjugates decreased. After about 4 h in the case of the 68 Ga-1 and 68 Ga-2 radioconjugates and about 4 days in the case of the 177 Lu-1 and 177 Lu-2 radioconjugates, the peaks corresponding to the tested radiopreparations were often hardly visible, indicating almost complete biodegradation of the radioconjugates. Radioactivity measurements of the precipitated protein serum components and the supernatants indicated that, in the case of the 68 Ga-1 and 177 Lu-1 radioconjugates, about 15% of the radiocompounds was bound to the protein serum components. In the case of the 68 Ga-2 and 177 Lu-2 radioconjugates, about 28% of the radiocompounds was bound with protein serum components.

Discussion
The aim of the present study was to synthesize and to characterize the physicochemical properties of radiolabeled A7R peptide and Lys(hArg)-Dab-Pro-Arg peptidomimetic for potential application as diagnostic or therapeutic receptor radiopharmaceuticals. According to the literature data, the heptapeptide A7R is known to be an effective antagonist of the VEGF-A 165 binding with NRP-1 and to show in vivo anti-angiogenic properties [35], whereas the Lys(hArg)-Dab-Pro-Arg peptidomimetic is much more active (IC 50 = 0.2 µM) than the heptapeptide A7R (IC 50 = 5.9 µM [42]), with a stability half-life in human serum that is nearly 2 days [41,43]. To be able to synthesize radioconjugates, we attached a DOTA chelator to the initial compounds-A7R or its shorter analogue-via an Ahx linker.
All the designed radioconjugates were synthesized with high radiochemical yield and high radiochemical purity (Figure 2 right), and the methods for their synthesis were relatively simple and cheap. Identity of the radioconjugates was confirmed by ESI-MS analysis of their cold reference compounds.
All radioconjugates turned out to be highly hydrophilic compounds with logP values in the range of −3.40 to −4.57 (Table 4). Moreover, the radioconjugates with Ga-68 turned out to be visibly less lipophilic due to a free carboxyl group of the DOTA chelator. These values were much lower than those preferred for radiopharmaceuticals, in the ranges of 1 to 4 and 1.5 to 2.5, which are suitable for crossing the blood-tissue [66] and blood-brain barriers [67], respectively. However, the ability to cross the blood-tissue barrier is not a crucial parameter in this case due to the presence of VEGF 165 in the endothelium, the single layer of squamous endothelial cells that lines the interior surface of blood vessels, and its ability to interact directly with blood.
Radioconjugates 68 Ga-1 and 68 Ga-2 and radioconjugate 177 Lu-2 were sufficiently stable in solutions acting as human body fluids, PBS buffer, and Cys or His solutions. However, the stability of the 177 Lu-1 radioconjugate that contained therapeutic radionuclide Lu-177 and based on the A7R peptide turned out to be rather insufficient. After 6 days of incubation, which is just one half-life of the radionuclide Lu-177, the radioconjugate decomposition products were already visible ( Figure 6). Because this is a potential therapeutic radiopharmaceutical containing radionuclide with a half-life of 6.65 days, such stability in these solutions is rather low.
Stability studies of the radioconjugates in human serum showed relatively rapid decomposition of all radiocompounds due to enzymatic biodegradation of A7R peptide and Lys(hArg)-Dab-Pro-Arg peptidomimetic ( Figure 8). Comparing the RP-HPLC radiochromatograms recorded after a 10 min incubation, it is apparent that the radioconjugates 68 Ga-2 and 177 Lu-2 were more readily biodegraded in HS compared to the radioconjugates 68 Ga-1 and 177 Lu-1. In the case of 177 Lu-1 radiopreparation after 4 days of incubation, the radioconjugate was still visible in the sample, while in the case of 177 Lu-2 after 1 day of incubation, only traces of radioconjugate were present in the sample. Moreover, during the incubation of 68 Ga-1 and 177 Lu-1 in HS, about 15% of the radiopreparations were bound to the serum peptide components, while in the case of radioconjugates 68 Ga-2 and 177 Lu-2, almost twice as much, about 28%. This difference can be explained by the less-polar nature of the A7R (Ala 1 -Thr 2 -Trp 3 -Leu 4 -Pro 5 -Pro 6 -Arg 7 -OH) peptide and the polar nature of the Lys 1 (hArg)-Dab 2 -Pro 3 -Arg 4 peptidomimetic. The former compound had three reactive functional groups-the guanidine group of Arg 7 , the amine group of Ala 1 , and the carboxyl group of Arg-while the peptidomimetic had six reactive functional groups-the three amino groups of Lys 1 , hArg, and Dab 2 , the two guanidine groups of hArg and Arg 4 , and one carboxyl group of Arg 4 . These reactive groups, depending on the pH and under the influence of external conditions, can assume, respectively, positive or negative charges, and interact with the protein components of the human serum. Despite the fact that one amine group was used to attach the DOTA chelator via the Ahx linker in both conjugates, the radioconjugates produced by the labeling reactions of conjugate 1 and 2 should still differ in charge, polarity, and chemical reactivity because of differences in the physicochem-ical properties of their parent compounds, the A7R peptide and Lys(hArg)-Dab-Pro-Arg peptidomimetic.
Based on the above data, it should be assessed that, unfortunately, radioconjugates 68 Ga-2 and 177 Lu-2 that were based on the more biologically active biomolecule, Lys(hArg)-Dab-Pro-Arg peptidomimetic, were less stable in HS than those based on the A7R peptide, 68 Ga-1 and 177 Lu-1. Such a low stability of 68 Ga-2 and 177 Lu-2 was initially surprising because, according to the literature data, the determined half-life of the Lys(hArg)-Dab-Pro-Arg peptidomimetic in HS was nearly 2 days (41 h), and the period of its complete decomposition in HS estimated from the graph was over 4 days [43]. As determined from our experiments, the period of almost complete enzymatic biodegradation of radioconjugate 177 Lu-2, resulting from enzymatic biodegradation of the Lys(hArg)-Dab-Pro-Arg peptidomimetic, was less than 1 day. These two seemingly contradictory results were due to the different conditions of the stability studies. In the first case, the peptidomimetic concentration in the sample was 0.9 µmol/mL, which allowed us to detect the biodegradation using the UV/VIS detection [43]. In the second case, the peptidomimetic concentration was equal to the concentration of the radioconjugate and was incomparably lower, i.e., about 2 nmol/mL; experiments with such a low radioconjugate concentration were possible due to using gamma detection. Consequently, in the first case, the test was carried out with saturation of the enzymes present in HS sample, while in the second case, the amount of the peptidomimetic was much smaller than the amount of enzyme. Hence, in the first case, intact peptidomimetic was visible in the sample for about 4 days, while this period was only 1 day in the present study.
To conclude, our study showed that all radioconjugates obtained in the presented work were not sufficiently stable in HS, and so they unfortunately do not fully meet the requirements for radiopharmaceuticals. Nevertheless, the structure of the peptidomimetics can be modified easily, which makes it possible to design a new formulation that is more stable in HS. In the near future, we plan to modify them by using D-amino acids or replacing one or more peptide bonds with their bioisosteres (e.g., N-methyl peptide bond, or reduced bond -CH 2 -NH-). For radiopreparations sufficiently stable in human serum and suitable for in vivo applications, we plan more advanced biological tests (using NRP-1 overexpressing cell lines) and finally tests on living organisms (a biodistribution study in mice or rats).